EPA/600/R-12/064 | December 2012 | www.epa.gov/ord
United States
Environmental Protection
Agency
Evaluation of
Reaerosolization of Bacillus
Spores from a Sod Matrix
Office of Research and Development
National Homeland Security Research Center
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DISCLAIMER
The United States Environmental Protection Agency, through its Office of Research and
Development's National Homeland Security Research Center, funded and managed this
investigation through EP-D-10-070 WA 0-06 Task 3 with Alion Science and Technology. This
report has been peer and administratively reviewed and has been approved for publication as
an Environmental Protection Agency document. It does not necessarily reflect the views of the
Environmental Protection Agency. No official endorsement should be inferred. The
Environmental Protection Agency does not endorse the purchase or sale of any commercial
products or services. This report includes photographs of commercially available products. The
photographs are included for purposes of illustration only and are not intended to imply that the
Environmental Protection Agency approves or endorses the product or its manufacturer.
Questions concerning this document or its application should be addressed to:
JackyAnn Rosati Rowe, Ph.D.
National Risk Management Research Lab
Office of Research and Development
U.S. Environmental Protection Agency (MD-E305-01)
109. T.W. Alexander Drive
Research, Triangle Park, NC 27711
e-mail: rosati.jacky@epa.gov
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ACKNOWLEDGMENTS
This effort was completed under U.S. EPA contract #ED-C-10-070 with Alion Science and
Technology. The support and efforts provided by Alion Science and Technology are gratefully
acknowledged.
Additionally, the authors would like to thank the peer reviewers for their significant contributions:
Dale Greenwell, US EPA ORD, Rebecca Connell US EPA OSWER, Marissa Mullins, US EPA
OSWER, and Marshall Gray, US EPA, ORD.
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TABLE OF CONTENTS
Disclaimer ii
Acknowledgments iii
Table of Contents iv
List of Tables v
List of Figures vi
Acronyms and Abbreviations vii
Executive Summary viii
1. Introduction 1
2. Facilities and Materials 2
2.1. Test Facility: Aerosol Wind Tunnel 2
2.2. Surface: Grass-Soil (Sod) Matrix 3
2.3. Spray Material and Apparatus 6
2.3.1. Bacillus thuringiensis var kurstaki (BtK) Material 6
2.3.2. Agricultural Sprayer 6
3. Sampling and Analytical Methods 8
3.1. Microbiological Sampling and Analysis 8
3.1.1. Saturation Sampler 8
3.1.2. Microbiological Assays 9
3.2. Ultraviolet Aerodynamic Particle Sizer (UV-APS) 9
4. Experimental Approach 13
5. Results and Discussion 15
6. Conclusions 19
7. References 20
8. Supporting Documentation 21
IV
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LIST OF TABLES
Table 1. Details of experimental conditions 14
Table 2. Fraction resuspended from high RH experiments 16
Table 3. Fraction resuspended from high RH experiments 16
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LIST OF FIGURES
Figure 1. Plan view of the aerosol wind tunnel 2
Figure 2. Schematic diagram of experimental setup with sod in the HETS 4
Figure 3. Healthy sod in trays in HETS before wind tunnel testing 5
Figure 4. Dried-out sod in trays in HETS after wind tunnel testing 5
Figure 5. Spore stain of the Biologica BtK spore suspension (spores are stained green,
and vegetative cells are red) 6
Figure 6. Saturation samplers downwind of sod in the HETS 8
Figure 7. UV-APS graph of particle size and fluorescence intensity for particles sampled
off of a filter loaded with BtK spore suspension 11
Figure 8. UV-APS graph of particle size and fluorescence intensity distribution for BtK
spore suspension spray in AWT 12
Figure 9. Fraction resuspended vs. time from high speed, high RH runs 17
Figure 10. Fraction resuspended vs. time from high speed, low RH runs 18
VI
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ACRONYMS AND ABBREVIATIONS
ATF
AWT
BtK
CPU
cm
CV
EPA
ft3
h
hi-vol
Hp
HETS
HSRP
in
Km
kPa
kW
L
m
m3
min
mph
nm
ORD
PBS
RH
rpm
s
SD
STS
TNTC
ISA
TSAB
UV-APS
Aerosol Test Facility
aerosol wind tunnel
B. thuringiensis var kurstaki
colony forming units
centimeter
coefficient of variance
U.S. Environmental Protection Agency
cubic feet
hour
high volume [sampler]
horsepower
human exposure test section
Homeland Security Research Program
inch
kilometer
kiloPascal
kilowatt
liter
meter
cubic meter
minute
milliliter
miles per hour
nanometers
Office of Research and Development
phosphate buffered saline
relative humidity
revolutions per minute
seconds
standard deviation
sampler test section
too numerous to count
tryptic soy agar
tryptic soy agar with 5% sheep's blood
Ultraviolet Aerodynamic Particle Sizer
micrometers
VII
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EXECUTIVE SUMMARY
The U.S. Environmental Protection Agency (EPA) has the responsibility for protecting human
health and the environment. To this end, the EPA Office of Research and Development (ORD)
Homeland Security Research Program (HSRP) is investigating the reaerosolization of Bacillus
anthracis in support of improving upon EPA's capabilities to respond to and recover from a wide
area release.
In this report, the investigation of reaerosolization of B. thuringiensis var kurstaki (BtK), a
simulant for B. anthracis, on a grass-soil matrix (sod) is presented. Sod (grass-soil matrix) was
sprayed using a solution containing BtK to investigate the fraction of spores resuspended from
the surface at two different humidity levels (30% and 70%). Filter-based sampling was used to
sample spores detached from the sod.
Resuspension was consistent after the initial (0 h) sample even though the grass-soil matrix
became desiccated during the experiment. The fraction resuspended from tests performed at
low RH (1.33 x 10"4) was an order of magnitude higher than from the high RH tests at the same
wind speed (1.95x 10"5).
VIII
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1. INTRODUCTION
A large outdoor release of 6. anthracis spores may result in spores being widely dispersed and
deposited on a wide range of surfaces (concrete, asphalt, soil, grass, etc.). In the 2001 6.
anthracis letter incidents, it was found that the spores that deposited on various indoor surfaces
reaerosolized (i.e., resuspended), which spread the contamination and exposed persons in
contaminated areas to inhalation hazards (Weis et al., 2002). In addition, spores adhered to
shoes and were tracked to many other buildings by evacuees and first responders.
Temperature, relative humidity, air movement, and physical disruption affect the amount of
reaerosolization and tracking from contaminated to uncontaminated areas. Research is needed
on how B. anthracis spores resuspend from various types of outdoor surfaces under varying
environmental conditions, as well as sampling methods to correlate surface deposition with
resuspension. Such research will aid in the assessment of exposure risk and mitigation
strategies.
The U.S. Environmental Protection Agency (EPA) has the responsibility for protecting human
health and the environment from such incidents through mitigation, consequence management,
and decontamination. To this end, the EPA Office of Research and Development (ORD)
Homeland Security Research Program (HSRP) is conducting research to investigate the outdoor
surface adhesion and reaerosolization of B. anthracis surrogate.
In this report, the investigation of reaerosolization of B. thuringiensis var kurstaki (BtK), a
simulant for B. anthracis, on a grass-soil matrix (sod) is presented. This work was done in the
controlled environment of an aerosol wind tunnel.
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2.
FACILITIES AND MATERIALS
2.1. Test Facility: Aerosol Wind Tunnel
To evaluate possible resuspension, an aerosol wind tunnel was used to provide a controlled
environment with well-defined velocity profiles. An overview of the Aerosol Test Facility (ATF)
wind tunnel, a facility run by the National Homeland Security Research Center (NHSRC), Office
of Research and Development (ORD), EPA, is shown in Figure 1. In plan view, the aerosol wind
tunnel (AWT) is rectangular in shape with outside dimensions of approximately 20 m by 14 m.
Flow through the recirculating wind tunnel during all operations is counterclockwise. There are
few flow obstructions, and several doors with locks allow access to all sections of the wind
tunnel. The human exposure test section (HETS) has a cross-section of 3.66 m wide by 3.05 m
high by 9 m long. The wind speed in the HETS can be varied from 0.1 to 2.22 m/s (0.36 to 8
km/h). At the sampler test section (STS), the wind tunnel cross-section is 1.75 m wide by 1.45 m
high by 6.1 m long. The wind speed in the STS can range from 0.56 to 13.3 m/s (2 to 48 km/h).
Each of the two test sections has its own movable traverse designed for three-dimensional
positioning of instruments with remote control and readout of position.
SAMPLER TEST SECnON
5.75»4.75x20FT
Figure 1. Plan view of the aerosol wind tunnel.
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The wind tunnel is of fixed geometry, and varying wind speeds are achieved by controlling the
volumetric flow rate. Major flow through the wind tunnel is provided by a direct- drive, adjustable-
blade, vane-axial fan (Twin City Fan and Blower, Minneapolis, MN) capable of providing
approximately 2002 m3/min (71,500 ft3/min) against 0.97 kPa (3.89 inches of water) pressure
drop at 1133 rpm at a power requirement of 56 kW (75 hp). This blower is capable of driving the
wind tunnel at speeds up to 48 km/h (30 mph) in the STS. Wind speed is controlled through a
variable-speed drive combined with a fan pitch system that regulates the rotational rate of the
fan.
Because high-speed operation of the wind tunnel adds a significant amount of heat to the
recirculating airstream, a cooling coil/chilled water system is used to control the tunnel
temperature. Controlled recirculation of chilled water through the cooling coil counteracts the
continued heat input and allows the wind tunnel to be operated at specified temperatures.
Humidity in the wind tunnel is maintained at 50% relative humidity (RH), but can be varied
according to test requirements. This is achieved by the combination of a desiccant dehumidifier
and a deionized water steam humidifier. After the humidity reaches the target condition, the
dehumidifier operates at a constant (low) setting and the humidifier output is automatically
controlled to maintain the target set point.
The wind tunnel includes a bank of high efficiency, mini-pleated filters downstream of the
sampler test section (Figure 1) to remove aerosols not collected by the samplers. This primary
filter bank effectively prevents the continuous accumulation of material in the tunnel interior,
dramatically reducing the background level of the test material in the airstream.
2.2. Surface: Grass-Soil (Sod) Matrix
The sod used for wind tunnel experiments was grown from Scotts Gold Standard Brand Pro
(Scotts Company, Marysville, OH) grass seed, a premium turf-type tall fescue. We investigated
the types of grass used on the National Mall in Washington, DC, and found that this was one of
the many types used throughout the year. Additionally, tall fescue is a widely grown cool-
season grass that is tolerant to heat and drought, is disease resistant, requires minimum care,
and is widely used across the southeastern United States.
The sod was placed in shallow trays in the HETS, 1.40 m x 1.78 m (cross wind x along wind),
0.84 m above the wind tunnel floor, as shown in Figure 2. The top of the grass blades was
approximately 0.92 m above the floor. The spray nozzles were 0.86 m above the sod surface.
The high-volume (hi-vol) samplers were located 1.35 m downwind of the downwind edge of the
sod. Figure 3 and Figure 4 show sod in the trays installed in the HETS before and after wind
tunnel testing.
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V
Honeycomb
Aerosol Test Facility Wind Tunnel Test Sections (plan view)
Human Exposure Test Section (NETS)
1.78m
Hi-Vol
samplers
Sod
1.35m
1.78m
Ill
O O O 0
H -Vol samplers
(front view)
Sampler Test Section (STS)
Honeycomb
Sod (side view)
Figure 2. Schematic diagram of experimental setup with sod in the HETS.
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Figure 3. Healthy sod in trays in HETS before wind tunnel testing.
Figure 4. Dried-out sod in trays in HETS after wind tunnel testing.
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2.3. Spray Material and Apparatus
2.3.1. Bacillus thuringiensis var kurstaki (BtK) Material
Biologies Process Development (Poway, CA) supplied BtK spores as a solid. The material was
resuspended in sterile distilled water. A serial dilution of the sample was performed to the 10"7
and a spore stain (see Figure 5) was performed. The sample was diluted in phosphate buffer
solution (PBS) and plated in triplicate on blood agar. The plates were incubated at 35 ± 2 °C for
24 hours. The concentration of the prepared stock was 1 x 107 colony-forming units per milliliter
(CFU/mL). The material from Biologies was used in all material testing. The BtK suspensions
were refrigerated at 2-8 °C.
Figure 5. Spore stain of the Biological BtK spore suspension (spores are stained green,
and vegetative cells are red).
2.3.2. Agricultural Sprayer
Material was dispersed using a modified Fimco (North Sioux City, SD) agricultural sprayer with a
12-volt Hypro 4 roller pump (model 2570-0013, New Brighton, MN). A sprayer boom designed to
spray from up to five nozzles was mounted on the movable traverse in the HETS. For these
experiments, the boom was set up with two Teejet TX4 Conejet nozzles (Wheaton, IL). When
operated at a line pressure of 30 psi, the nozzles produced a droplet size with median volume
diameter of 140 urn. The two spray nozzles were 25 inches apart. The flow rate through each
nozzle was measured using a timed catch. Material from the spray nozzles was captured in
Class A graduated cylinders for a measured length of time. The average flow rate was reported
for each experiment in milliliters per minute (mL/min).
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The line from the sprayer to the spray nozzles mounted on the wind tunnel traverse was 1615
cm long with an inner diameter of 1.3 cm (0.5 in). The volume of the sprayer line was 2045 ml_.
The sprayer and line were decontaminated between runs with a 2% bleach solution and rinsed
with sterile water. The line was then completely purged. Immediately before each spray run, the
sprayer was loaded with the new spray material and it was run with the spray nozzles enclosed
in buckets to charge the sprayer line with material before spraying the test surface.
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3.
3.1.
SAMPLING AND ANALYTICAL METHODS
Microbiological Sampling and Analysis
3.1.1. Saturation Sampler
A saturation sampler was designed using four hi-vol sampling pumps (see Figure 6). The pumps
were placed on a rack with the filters centered 0.92 m above the floor and were operated at a
flow rate of 1200 L/min each. The flow rates for each pump were measured using a BGI
(Waltham, MA) high-volume calibrator. Autoclavable 102-mm polyester-fiber filters (National Air
and Radiation Environmental Laboratory [NAREL], Office of Radiation and Indoor Air [ORIA],
EPA, Montgomery, AL) were used to collect airborne particles. The collection efficiency of the
filters has been shown to be 99.8% at flow rates of IOOO and 1333 L/min for particles with
diameters of 0.5 urn and larger (Lovelace Respiratory Institute, 2005).
Figure 6. Saturation samplers downwind of sod in the HETS.
The extraction efficiency for the experiments discussed in this report was determined in our
laboratory to be 9% (±1.9%). A correction factor to compensate for the low extraction efficiency
was applied to the collected data. Extraction efficiency was determined by inoculating a known
concentration of material onto each of three sterile filters. The filters were allowed to dry in a
biosafety cabinet for 30 min. The filters were then placed into sterile 250 mL wide-mouth bottles
containing 30 mL of PBS with Trition X-100. Filters were vortexed for 30 seconds and then
shaken by hand for 10 seconds before the solution was inoculated on to tryptic soy agar (TSA)
plates in triplicate. Plates were incubated at 35 ± 2 °C for 24 hours. The plates were manually
8
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counted and reported as colony-forming units per milliliter (CFU/mL). Despite the low extraction
efficiency, this sampling method is the best available to us at this time.
3.1.2. Microbiological Assays
The BtK spores were obtained as a solid from Biologies, which was resuspended in sterile
distilled water, and the BtK spore suspension was refrigerated at 2-8 °C. The concentration of
the stock was 1x107 or higher CFU/mL. Pre-poured TSA plates were purchased and certified by
the manufacturer as being free of contamination through a certificate of analysis. These plates
were visually inspected prior to use, and any plates that were contaminated were discarded.
Liquid from the sampler impingers, swabs, and/or filters were diluted using 10-fold serial
dilutions to achieve a plate count between 30 and 300 CPU and inoculated onto TSAITSAB
plates. The plates were incubated for 24 hours at 35 ± 2 °C. The colonies of BtK are rough, dull,
and round with entire margins. They were manually counted and reported as CFU/mL. Gram
stain analysis was performed to ensure that the BtK was positively identified. To verify that a 24-
hour incubation time was sufficient, a subset of plates was counted after 24 hours, incubated for
an additional 24 hours, and recounted. There was no significant increase in the number of
colonies from the 24-hour to the 48-hour plates.
A positive and a negative control were performed for each test batch. A positive control
consisted of plating the spore suspension prepared for the sprayer to determine the
concentration of spores that were being sprayed. A negative control of the phosphate buffer was
prepared by plating 0.5 mL onto an agar plate and spreading evenly by rocking the plate.
All colonies were enumerated on countable plates (30-300 CPU). Plates with less than 30CFU
were considered to be negative. Plates with greater than the countable range were considered
too numerous to count (TNTC) and were reprocessed with diluted sample to achieve countable
plates. Results were recorded to two significant figures. Triplicate plates were prepared for the
dilutions that were plated. The plates were counted and the average per plate was calculated
using the following equation:
CPU nCFU^ + #CFU2 + #CFU3 dilution factor
= x
mL 3 volume plated
3.2. Ultraviolet Aerodynamic Particle Sizer (UV-APS)
The UV-APS (TSI model 3314, Shoreview, MN) is a particle spectrometer that measures the
aerodynamic diameter, the scattered light intensity, and the fluorescence of airborne particles.
The fluorescence measurement is based on excitation illumination at a fixed wavelength of 355
nm and fluorescence emission in the wavelength region 420-575 nm. This measurement
provides information about the particle composition such as biological content or the presence of
other materials with known fluorescence characteristics. The UV-APS also provides count and
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mass size distributions and light scattering for particles with aerodynamic diameters from 0.5 to
20 urn
A UV-APS data-processing spreadsheet was developed to easily and accurately process large
amounts of data from the UV-APS. It is a Microsoft Excel workbook that contains Visual Basic
programs to help in the analysis of data from a file exported from the UV-APS data acquisition
software. The control workbook has two main functions: The first function imports the UV-APS
data from an Excel-ready ASCII file and loads multiple sample runs into separate sheets in the
data workbook. The second function of the control workbook calculates the total particle number
concentration in the desired particle size and fluorescence range for each sample. This is done
by setting the filter ranges in the control workbook for the particle size and fluorescence of
interest and pressing the Filter Totals button.
Figure 7 contains a three-dimensional graph showing the distribution of aerodynamic particle
size and fluorescence intensity for a 5-second sample with the UV-APS sampling directly off of a
filter loaded with dried BtK spore suspension. The figure shows a significant number of
fluorescent particles between 2.5 and 14 urn in diameter, representing individual spores and
agglomerates, with varying degrees of fluorescence intensity, as well as a large concentration of
background (non-fluorescent) particles. Figure 8 contains a similar graph for a 60-second
sample with the UV-APS sampling in the AWT while the test surface was being sprayed with
BtK spore suspension. A large number of fluorescent particles were sampled between 2 and 7
urn in diameter over the range of fluorescence intensity, along with a large concentration of
background (non-fluorescent) aerosol particles.
10
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Figure 7. UV-APS graph of particle size and fluorescence intensity for particles sampled
off of a filter loaded with BtK spore suspension.
11
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Figure 8. UV-APS graph of particle size and fluorescence intensity distribution for BtK
spore suspension spray in AWT.
12
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4. EXPERIMENTAL APPROACH
The experimental conditions for the test runs are documented in Table 1. The procedure for
each of the test runs was as follows:
1. Decontaminate the wind tunnel with the Steris H202 system (usually done the Friday before
the run began).
2. Set up the grass in the trays in the HETS and water until soil surface is damp (usually done
on Sunday, the day before the run began).
3. On Monday morning, day 1 of the test run, run a 1-hour sample before spraying at the test
wind speed to measure the background.
4. Stop the tunnel by setting the wind speed to 0.0 m/s. The time stopped is approximately 3
min. when the tunnel is stopped to deploy or collect filters for each sample, as noted here and in
the steps below,
5. Remove the filters from the pre-spray run, load new filters, and cover samplers.
6. Load spray material into the sprayer, charge the spray lines, and spray the grass with one
sweep of the sprayer traverse (traverse sweep take approximately 2.5 min).
7. Remove the spray nozzles and cover fittings on the traverse.
8. Allow 5-min drying time.
9. Turn the tunnel up to test wind speed for 5 min to purge.
10. Stop the tunnel and pull the covers off the filters.
11. Turn the tunnel up to test wind speed and run a 1-hour-long sample (designated as 0 h
sample).
12. Stop the tunnel and collect the filters.
13. Turn the tunnel up to test wind speed.
14. At 2 hours from the beginning of the test, stop the tunnel and load new filters.
15. Turn the tunnel up to test wind speed, wait 5 min (to purge material stirred up from walking
in the test section), and run a 1-hour-long sample (designated as 2 h sample).
16. Stop the tunnel and collect the filters.
17. Turn the tunnel up to test wind speed 18. Repeat steps 14-17 at 6, 24, 48, and 72 hours
from the beginning of the test.
13
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Table 1. Details of experimental conditions
HS=High Wind Speed; HRH= High RH; LRH=Low RH
Test
Description
Lie MRH
Run#1
(Turf 3)
HS HRH
Run #2
(Turf 4)
HS LRH
Run #1
(Turf 1)
HS LRH
Run #2
(Turf 2)
HS LRH
Run #3
(Turf 5)
WT Velocity
(mis)
HETS
2.2
2.1
2.3
2.3
2.1
STS
9.9
9.3
9.9
10.0
9.3
RH
(%)
69.9
70.3
30.0
30.2
30.3
Temp
(°C)
22.1
22.2
24.3
21.5
21.8
Drying/Purge
Time
Dry 5 min
Purge 5 min
Dry 5 min
Purge 5 min
Dry 5 min
Purge 5 min
Dry 5 min
Purge 5 min
Dry 5 min
Purge 5 min
Amount
Sprayed
3.0 x1011
5.6 x1011
3.5 x1011
2.4 x1011
4.7 x1011
Sampler
Types
Used
4 hi-vol
UV-APS
4 hi-vol
UV-APS
4 hi-vol
UV-APS
4 hi-vol
UV-APS
4 hi-vol
UV-APS
Sampling
Time
(min)
60
60
60
60
60
14
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5. RESULTS AND DISCUSSION
For these experiments, it was determined that the most appropriate quantity to calculate was the
fraction of material resuspended for each sampling interval. All calculations were executed in
Microsoft Excel spreadsheets. After the spreadsheets were completed, they were reviewed for
quality assurance to ensure accurate data transcription and calculation.
As stated in section 3.1.1 and 3.1.2, the samples collected on the hi-vol filters were extracted
and plated in triplicate. The fraction resuspended was calculated from these hi-vol filter results.
The total CPU collected on each filter was calculated as:
CFUaveraae x dilution factor x extraction volume
Total CPU collected = ^^ -^
volume plated
Where: CFUaverage was the average of the triplicate CPU counts for the filter, and the extraction
volume and volume plated were in milliliters (ml_). The air concentration, C, was then calculated
in units of CFU/Las:
Total CPU collected
C =
sampling flow rate x t x filter extraction efficiency
where the flow rate was in liters per minute (L/min), t was the sampling time (min), and the filter
extraction efficiency was experimentally determined in our laboratory (see Section 3.1.1 ). Next
the total amount resuspended was calculated by:
Total amount resuspended = C xtxux Amixed x 1000 L/m3
where u was the wind speed in the test section (m/s), and Amixed was the cross-sectional area of
the test section that was estimated to be well mixed and represented by the measured
concentration. For all of the experiments described, Amixed was 1.36 m2 (16% of the HETS height
x 75% of the HETS width), which was determined from flow data in the HETS. The fraction
resuspended was then calculated by:
Total amount resuspended (CPU)
Fraction resuspended = — —
Total amount sprayed (CPU)
where the total amount sprayed was the concentration in the sprayer (CFU/mL) times the
volume sprayed (ml_).
Table 2 and Table 3 present the calculated fraction resuspended for each experimental run
described above, along with the average, standard deviation (SD), and coefficient of variation
(CV). The results are plotted in Figure 9 and Figure 10 as fraction resuspended versus time for
high RH and low RH tests. The red line on each graph is the average of the data sets shown.
The fraction resuspended did not vary significantly over the test duration, although the initial (0
15
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h) sample was consistently higher than subsequent samples, as can be seen by the relatively
flat lines representing the average fraction resuspended over time in the figures. The average
fraction resuspended of all samples taken at high RH was 1.95x10"5, and the average at low RH
was 1.33 x 10"4, an order of magnitude greater than at high RH. The sod was watered when it
was placed in the trays in the wind tunnel and was green and healthy when the tests began (see
Figure 3). The grass and soil dried out during the course of the 72-hour long tests, especially at
low humidity (see Figure 4). This allowed for comparison of resuspension from healthy sod and
dried out sod, as is commonly found in the southern United States during the hot summer
months. As can be seen from the relatively constant amount of material resuspended after the
initial (0 h) sample (red lines in Figure 9 and Figure 10), the condition of the grass-soil matrix did
not have a significant impact on the amount of material that was resuspended.
Table 2. Fraction resuspended from high RH experiments.
HS HRH
Run#1
(Turf 3)
HS HRH
Run #2
(Turf 4)
Average
SD
CV
Oh
7.09x1Q-5
1.69x10'5
4.39x10'5
3.82x10'5
87%
2h
1.99x1Q-5
1.45x10'5
1.72x10'5
3.82x10'6
22%
6h
4.30x1Q-5
1.28x10'5
2.79 x10'5
2.14 x10'5
77%
24 h
1.07x1Q-5
4.96 x10'6
7.80 x10'6
4.03 x10'6
52%
48 h
1.49x1Q-5
6.12 x10'6
1.05x10'5
6.18 x10'6
59%
72 h
1.34x1Q-5
5.98 x10'6
9.70 x10'6
5.27 x10'6
54%
Table 3. Fraction resuspended from low RH experiments.
HS LRH
Run#1
(Turf 1)
HS LRH
Run #2
(Turf 2)
HS LRH
Run #3
(Turf 5)
Average
SD
CV
Oh
9.96x10'5
4.27 x10'4
3.20 x10'4
2.82 x10'4
1.67x1Q-4
59%
2h
6.65 x10'5
2.11 x10'4
8.59x10'5
1.2x10'4
7.85x1Q-5
65%
6h
7.79x10'5
1.78x10'4
5.62 x10'5
1.04x10'4
6.51 x ID'5
63%
24 h
8.06 x10'5
1.94x10'5
9.25 x10'5
1.22x10'4
6.24 x10'5
51%
48 h
2.52 x10'5
2.71 x 10'4
3.32 x10'5
1.10x10'4
1.39x1Q-4
127%
72 h
1.42x10'5
9.09 x10'5
7.12 x10'6
5.88 x10'6
3.98 x10'6
68%
16
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• TurfS
Turf4 Average
"S 0-01 ~
•o
I
w
a)
tt
c
o
LL
^
O
•o
&
at
F
V^^-
4 A A
O5O5O5O5O5O5O5C
T- CM CO ^ LO CD h
Time (hours)
Figure 9. Fraction resuspended vs. time from high speed, high RH runs.
17
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» Turfl A Turf2 • TurfS Average
•o 0.01 -
•D
C
0)
n
I
= 0.0001 '
^
3
I
1
0)
K
ip.in
^4_ A A _
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*
Time (hours)
Figure 10. Fraction resuspended vs. time from high speed, low RH runs.
18
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6. CONCLUSIONS
To evaluate resuspension of spores from a sod matrix, an aerosol wind tunnel (AWT) was used
to provide a controlled environment with well-defined velocity profiles. Sod (soil-grass matrix)
was placed inside the AWT and sprayed using BtK solution to investigate the fraction of spores
resuspended from the surface at two different humidity levels. In the HETS section of the AWT
where the sod was positioned, the ambient temperature was held at 23°C, wind speed was
approximately 2.2 m/s, and RH was held at either 30% (low) or 70% (high). A UV APS as well
as filter-based air sampling was used to determine the quantity of spores detached from the sod.
A resuspension fraction was determined based on the quantity of spores detached from the sod
matrix and the quantity of spores sprayed onto the sod matrix.
The resuspension fraction of BtK spores was consistent for 72 hours after the initial (0 h) sample
even though the grass-soil matrix became desiccated during the experiment. The fraction of
spores resuspended from tests performed at low RH (1.33 x 10"4) was an order of magnitude
higher than those resuspended from the high RH tests at the same wind speed (1.95 x 10"5).
This was likely because a greater force is required to detach the spores under high RH
conditions than under low RH conditions, probably due to increased capillary forces at the
higher RH. Additional work using atomic force microscopy (AFM) is being performed to confirm
this detachment force requirement.
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United States
Environmental Protection
Agency
PRESORTED STANDARD
POSTAGE & FEES PAID
EPA
PERMIT NO. G-35
Office of Research and Development (8101R)
Washington, DC 20460
Official Business
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